Mycobacterium tuberculosis is one of the most successful pathogens known today, killing millions of individuals worldwide every year. A hallmark of M. tuberculosis infection is that following phagocytosis the microorganisms resist lysosomal delivery, instead residing within phagosomes that do not fuse with lysosomes. Phagolysosomes are equipped with the machinery to generate peptide—MHC II complexes. Inhibition of phagolysosome fusion has been proposed to represent a mechanism by which M. tuberculosis escapes efficient antigen presentation by host MHC II complexes. In addition, cross-presentation of peptides derived from particulate antigens and exploiting the conventional MHC I pathway can occur via a putative phagosome-to-cytosol mechanism. Alternatively, this can occur by fusion and fission of phagosomes with endoplasmic reticulum-derived vesicles containing newly synthesized MHC I molecules.
BCG is a live attenuated vaccine derived from M. bovis back in 1919. More than three billion doses of the vaccine have been administered worldwide. Although BCG is relatively safe and inexpensive, its efficacy is highly variable (Fine, P. E. M., Lancet 346, 1339-1345, 1995). The reasons for the varying efficacy of BCG in protection against tuberculosis are poorly understood. One explanation builds on the observation that BCG has lost important genes during the laboratory attenuation process (Behr et al., Nature 389, 133-134, 1997). A great deal of work has been done to improve—with varying success—the efficacy of BCG by introducing additional copies of existing genes (Horwitz et al., PNAS, USA 97, 13853-13858, 2000) or by reintroducing some of the genes that were lost during the in vitro attenuation process (Pym et al., Nat. Med. 9, 533-539, 2003).
Alternative live vaccination strategies focus on attenuated M. tuberculosis. It is commonly assumed that tuberculosis disease will protect, at least partially, against subsequent reinfection. However, the failure of natural disease to protect against re-infection disease at a later point indicates that immunity evoked by natural infection is limited, partially explaining the relative ineffectiveness of vaccination with BCG. The limited post-infectious immunity may also indicate that M. tuberculosis actively escapes immune surveillance.
Recently, the present inventors reported that a putative mycobacterial zinc metallo-protease, Zmp1, may play an important role in disease pathogenesis by interfering with two pathways of pathogen defense: inflammasome activation and phagosome maturation (Master et al., Mycobacterium tuberculosis prevents inflammasome activation. Cell Host Microbe 3, 224-232, 2008).
It is the object of the present invention to provide a live mycobacterium-based medicament with increased immunogenicity and improved protective efficacy.
This object is solved by the use of at least one live mycobacterium of the M. tuberculosis complex for preparing a medicament, wherein the function of the zmp1-gene is at least partially inactivated, preferably inactivated.
It was found that live mycobacteria, wherein the function of the zmp1-gene is at least partially inactivated, preferably substantially or fully inactivated, elicit an increased immunogenicity and protective efficacy when compared to zmp1 active mycobacteria.
The term live mycobacterium of the M. tuberculosis complex as used herein refers to a mycobacterium species or strain which is a member of the M. tuberculosis complex, which includes but is not limited to M. tuberculosis, M. bovis BCG, M. bovis, M. africanum and M. microti. 
Preferably, a mycobacterium of M. bovis, preferably M. bovis BCG, or M. tuberculosis is used for preparing a medicament according to the invention.
The mycobacterium used for the invention is alive, i.e. capable of propagation in a host, in particular in a mammalian host, preferably in a human host.
It is self-evident, that live mycobacteria for medical use must be attenuated to a degree not harmful to patients in need thereof. Hence, the mycobacterium used for the invention is preferably non-virulent, i.e. the genes responsible for virulence have been inactivated, and does not evoke or at least evokes minor disease symptoms of a mycobacterial infection in a mammal, preferably human.
Mycobacteria of the M. tuberculosis complex produce endogenous antigens which are cross-reactive with M. tuberculosis. Antibodies raised against such cross-reactive antigen will also bind specifically to one or more antigens from M. tuberculosis and are capable of evoking and/or potentiating an immune response against M. tuberculosis infection in a mammal. For example, cross reactive antigens of M. bovis BCG will evoke and/or potentiate an immune response against M. tuberculosis. 
Mycobacteria of the M. tuberculosis complex are not only useful for expressing cross-reactive antigens but also have applications as a delivery system for the expression of exogenous or foreign antigens and/or immunogens. The efficacy of this delivery system lies in the long persistence in the immunized host (Stover et al., Nature, 351, 456-460, 1991; Aldovini and Young, Nature, 351, 479-482, 1991).
Exemplary suitable antigens for delivery via mycobacteria of the M. tuberculosis complex include viral, protozoal, tumour cell-derived, bacterial and fungal antigens. For example, antigens derived from H. pylori, measles virus, mumps virus, rubeola virus, B. burgdorferi (e.g. OspA), herpes virus, papilloma virus, Pneumococcus spp (e.g. surface protein A), tumour cells, leishmania (e.g. surface proteinase gp63), HIV or SIV may be used. Such an antigen may be useful in the treatment of ulcers, measles, mumps, rubeola, lyme disease, herpes, cancer, tetanus, diphtheria, leishmaniasis or AIDS.
In a preferred embodiment, mycobacteria for use in the present invention may also comprises genetic material encoding an antigen and/or immunogen exogenous or foreign to the mycobacterium. More preferably, the exogenous or foreign antigen and/or immunogen is selected form the group consisting of viral, protozoal, tumor cell-derived, bacterial and fungal antigens and immunogens, preferably selected from the group consisting of antigens and/or immunogens from H. pylori, measles virus, mumps virus, rubeola virus, B. burgdorferi, preferably protein ospA of B. burgdorferi, herpes virus, papilloma virus, Pneumococcus spp, preferably protein A of Pneumococcus spp., tumour cells, leishmania, preferably surface proteinase gp63 of leishmania, HIV and SIV.
Hence, the mycobacterium for use in preparing a medicament according to the invention is useful for the prophylaxis and/or treatment of diseases or medical conditions affected by antigen and/or immunogen expression of the mycobacterium. An antigen evokes antibody production in a host, an immunogen affects the immune system of a host.
Most evident as well as most preferred, the medicaments prepared from zmp1-inactivated mycobacteria according to the invention are useful for the prophylaxis and/or treatment of mycobacterial infections, preferably an infection of M. tuberculosis. 
In a further preferred embodiment the present invention is directed to the use according to the invention for the prophylaxis and/or treatment of a disease or medical condition selected from the group consisting of ulcers, measles, mumps, rubeola, lyme disease, herpes, cancer, tetanus, diphtheria, leishmaniasis and AIDS.
One or more live mycobacteria species and/or strains with an at least partially inactivated zmp1-gene function can be used for preparing a medicament according to the invention.
The term “function of the zmp1-gene” as used herein is intended to mean the function of the putative mycobacterial zinc metalloprotease Zmp1 in inflammasome activation, caspase-1-dependent activation and secretion of IL-1β as well as phagosome maturation in mammalian, in particular human macrophages.
Hence, the function of the zmp1-gene in a mycobacterium can be directly verified and/or quantified by investigating inflammasome activation, caspase-1-dependent activation and/or secretion of IL-1β and/or phagosome maturation in mammalian, in particular human macrophages upon infection with the mycobacterium of interest. Of course, other direct and indirect effects of protein Zmp1, e.g. effects resulting from IL-1β activation and/or secretion, are also indicative of the function of the zmp1 gene and may also be used instead of or in addition to the above direct indicators.
The term “at least partially inactivated”, as used herein, is meant to indicate a loss of zmp1 gene function directly and/or indirectly attributable to the expression of the zmp1 gene. Of course, the inactivation and extent of inactivation can best be determined in direct comparison to a mycobacterium of the same strain under identical experimental conditions but comprising a fully functional and expressed zmp1 gene. Preferably, “at least partially inactivated” relates to a loss of more than 10%, preferably more than 50%, more preferably more than 70%, most preferably more than 90% of a function directly and/or indirectly attributable to the expression of the zmp1 gene in a mycobacterium.
Partial and/or full inactivation of genes in mycobacteria of the M. tuberculosis complex for use in the present invention can be achieved by any conventional method in the art of gene mutation, e.g. recombinant insertion, replacement, deletion, frameshift and/or homologous recombination. Suitable specific methods for partially and/or fully inactivating genes, in particular the zmp1 gene in mycobacteria are described in WO 02/50262 (recA mutants), Master et al., supra, and the examples below.
It was found that the threshold for the induction of an immune response to M. tuberculosis antigens in immunized mammals was ten times less for mammals immunized with zmp1-deficient mycobacterial strains when compared to the threshold necessary for inducing the immune response in mammals immunized with the corresponding wild type, i.e. zmp1 activated, mycobacterial strain. Consequently, medicaments prepared from mycobacteria according to the invention evoke a substantially increased immunogenicity associated with increased proliferative and cytokine secreting immune responses.
Furthermore, the protective efficacy of mammals immunized with a medicament, i.e. pharmaceutical composition/vaccine, prepared according to the invention against M. tuberculosis challenge was demonstrated to be clearly superior over the protective efficacy achieved in mammals immunized with the corresponding wild type mycobacterial strain. In comparison, mammals immunized according to the invention demonstrated upon M. tuberculosis challenge a more pronounced and more frequent lymphocytic infiltration into lungs.
Last but not least, increased immunogenicity and protective efficacy of medicaments prepared according to the invention manifests itself in a substantially prolonged mean survival time of immunized mammals upon challenge with virulent M. tuberculosis. 
Consequently, the medicaments prepared according to the invention present a significant improvement for therapeutic, prophylactic and other medical or veterinary applications of mycobacteria for delivering endogenous, exogenous and/or foreign antigens and/or immunogens to a mammal in need thereof.
A second aspect of the present invention concerns pharmaceutical compositions comprising as active substance at least one live mycobacterium of the M. tuberculosis complex, wherein the function of the zmp1-gene is at least partially inactivated, preferably inactivated, optionally and preferably combined with pharmaceutically acceptable conventional excipients and/or carriers.
A pharmaceutical composition according to the present invention is any composition, preferably a vaccine, formulated for direct medical administration to a patient in need thereof, preferably a mammal, more preferably a human.
Preferably, the mycobacterium in the pharmaceutical composition is M. bovis, preferably M. bovis BCG, or M. tuberculosis. It is also preferred that the mycobacterium used is non-virulent.
In a preferred embodiment the pharmaceutical composition according to the invention is one, wherein the mycobacterium comprises genetic material encoding an antigen and/or immunogen exogenous or foreign to the mycobacterium, wherein the antigen and/or immunogen is preferably selected form the group consisting of viral, protozoal, tumor cell-derived, bacterial and fungal antigens and immunogens, more preferably selected from the group consisting of antigens and/or immunogens derived from H. pylori, measles virus, mumps virus, rubeola virus, B. burgdorferi, preferably protein ospA of B. burgdorferi, herpes virus, papilloma virus, Pneumococcus spp, preferably protein A of Pneumococcus spp., tumour cells, leishmania, preferably surface proteinase gp63 of leishmania, HIV and SIV.
Preferably, the pharmaceutical composition according to the invention is one suitable for the prophylaxis and/or treatment of a disease or medical condition affected by antigen and/or immunogen expression of the mycobacterium, more preferably the prophylaxis and/or treatment of mycobacterial infections, preferably an infection of M. tuberculosis, also more preferably for the prophylaxis and/or treatment of a disease or medical condition selected from the group consisting of ulcers, measles, mumps, rubeola, lyme disease, herpes, cancer, tetanus, diphtheria, cancer, leishmaniasis and AIDS.